Dual-energy x-ray absorptiometry (DXA) provides a measure of lean soft tissue (LST). LST hydration, often assumed to be constant, is relevant to several aspects of DXA body composition estimates. The aims of this study were: to develop a theoretical model of LST total body water (TBW) content and to examine hydration effects with empirically-derived model coefficients; and then experimentally test the model's prediction that, in healthy adults, LST hydration is not constant but varies as a function of extra- and intra-cellular water distribution (E/I). The initial phase involved TBW/LST model development and application with empirically derived model coefficients. Model predictions were then tested in a cross-sectional study of 215 healthy adults; LST was measured by DXA, extracellular water (ECW) by NaBr dilution, intracellular water (ICW) by whole-body ⁴⁰K counting, and TBW by 2H2O dilution. TBW estimates calculated as ECW + ICW were highly correlated with (r = 0.97, SEE = 2.1 l, P < 0.001) and showed no significant bias compared to TBW measured by ²H₂O. Model-predicted TBW/LST was almost identical to experimentally-derived values (X±SD) in the total group (0.767 vs. 0.764 ± 0.028). LST hydration was significantly correlated with E/I (total group, r = 0.30, SEE = 0.027, P < 0.001). While E/I increased with age (men, r = 0.48; women, r = 0.37, both P < 0.001), the association between TBW/LST and age was non-significant. Hydration of the DXA-derived LST compartment is thus not constant but varies predictably with extra and intracellular water distribution. This observation has implications for the accuracy of body fat measurements by DXA and the use of TBW as a means of checking DXA system calibration.